1. Field
The present invention relates to reagents and methods for detecting a molecule of interest in a biological sample. More particularly, the present invention relates to antibody conjugates and methods for using such conjugates to detect a molecule of interest in a biological sample such as a tissue section.
2. Background
Covalent conjugates of antibodies and signal-generating moieties can be used in immunoassays for detecting specific target molecules in biological samples. The antibody portion of such conjugates specifically binds to a target in the sample and the signal-generating moiety is utilized to provide a detectable signal that indicates the presence/and or location of the target. One type of conjugate that has become widely used, especially for immunohistochemical analysis, is a conjugate of an antibody and an enzyme (antibody-enzyme conjugate). A detectable signal is generated by adding a substrate to the sample and the enzyme portion of the conjugate converts the substrate to, for example, a colored, fluorescent or luminescent product at the site where the antibody portion is bound to its target.
Antibody-enzyme conjugates are typically prepared using polyfunctional (typically bifunctional) coupling reagents that are characterized by having at least two reactive groups, one of which is reacted with a functional group on the antibody and the other of which is reacted with a functional group on the enzyme. However, coupling can lead to inactivation of either or both of the antibody and the enzyme due to steric effects or because the coupling reagents react with functional groups located on portions of the enzyme or antibody that are critical for their function or specificity.
An approach to minimizing loss of antibody specificity and enzyme activity is to use a coupling scheme that is specific to particular amino acid residues on either or both of the antibody and the enzyme that are not associated with their functions. This approach is exemplified by the method for Fc-specific conjugation as described in U.S. Pat. No. 5,191,066, which is incorporated by reference herein. In this method, sulfhydryl groups (thiol groups) are introduced specifically to a glycosylated region of the Fc portion of an antibody and used along with a linker molecule to covalently attach an enzyme to the antibody. Since the Fc portion is not involved with the specific binding properties of the antibody, such conjugates retain greater specificity, which increases the detectable signal for a particular target molecule of interest and lowers background due to non-specific binding.
Although site specific conjugation can be used to help minimize loss of antibody specificity and enzyme activity due to loss of critical functional groups, such methods do not address loss of antibody specificity and enzyme activity that arise from steric effects such as those steric effects due to aggregation of multiple conjugates and from interactions between the antibody and the enzyme(s) in a conjugate. Detrimental steric effects also can arise due to unintended cross-linking between multiple enzymes, antibodies and/or conjugates, which occurs during preparation of a conjugate composition.
One approach to minimizing loss of antibody specificity and enzyme activity due to steric effects is to increase the length of the coupling reagent in order that the antibody and enzyme are separated by a greater distance. This approach is exemplified by the methods and conjugation reagents disclosed in U.S. Pat. No. 5,053,520. In this method, heterobifunctional linkers having extended alkyl, cycloalkyl, alkyl-cycloalkyl and aromatic portions are used to couple an antibody to an enzyme(s). Although such linkers contain more atoms and should provide greater separation between an antibody and an enzyme(s), it is believed that the hydrophobic nature of such linkers increases detrimental aggregation of conjugates in aqueous solution due to hydrophobic effects. In addition, such linkers are flexible enough to permit detrimental intra-conjugate interactions between the antibody and the enzyme(s) as a conjugate collapses in on itself to minimize its size due to hydrophobic effects.
An attempt to minimize detrimental aggregation between conjugates is described in U.S. Pat. No. 4,810,638, which describes the use of homo-bifunctional, bis-maleimidopolyalkylene glycol linkers to prepare antibody-enzyme conjugates. However, use of such homo-bifunctional linkers can lead to cross-linking of antibodies, enzymes and/or conjugates during preparation of the conjugates. Cross-linking increases the average size and counteracts to some extent the increased water solubility imparted by using the glycol linker. Furthermore, cross-linking leads to lower monodispersity in a conjugate composition, which can have detrimental effects on consistency of results, especially in tissue and cell samples where detection of a target with a conjugate may be limited by diffusion through cell membranes.
Some heterobifunctional polyethylene glycol linkers are known, but there are no known attempts to use them as coupling reagents for forming antibody-enzyme conjugates. Rather, as disclosed in Chen et al. (Chen et al., “The use of bifunctional polyethylene glycol derivatives for coupling of proteins to and cross-linking of collagen matrices,” J. Mater. Sci. Mater. Med., 13: 1029-1035, 2002), such agents have been utilized to prepare degradable matrices to which active proteins are linked for the purposes of tissue engineering.
From the standpoint of increasing the signal generated by a given antibody conjugate it is desirable to conjugate multiple enzymes to a single antibody. However, as the number of enzymes linked to a single antibody increases, the likelihood increases that conjugate function will be impaired for steric reasons due to crowding of multiple enzymes around the single antibody. One approach to minimizing crowding of enzymes is to employ a scaffold to provide separation between enzymes and between enzymes and antibodies or antibody fragments. U.S. Pat. Nos. 6,252,053 and 6,613,564, for example, describe the use of polylysine or dextran scaffolds to increase separation between enzymes, while still effectively increasing the number of enzyme molecules per specific binding component [specifically F(ab′)2 fragments]. While the approach described in these patents does increase the average number of signal-generating moieties per specific-binding component, the use of a polymeric scaffold (typically of low mono-dispersity) increases background and decreases reproducibility. The high molecular weight (typically greater >1 MDa) of such constructs can hinder diffusion and tissue/cell penetrability is diminished, thereby reducing signal.
What is needed, therefore, is an antibody/signal-generating conjugate composition that overcomes at least the described limitations of prior approaches. In particular, antibody conjugates of enzyme (and methods of making the same) that are smaller and yet retain the high signal generating capacity of larger scaffolded conjugates are desirable.